Abstract Inorganic phosphate (PO43-/Pi) is a critical regulator of the physiologic biomineralization process in skeletal and dental tissues, and is a major contributing factor to pathologic mineralization of blood vessels. The central role of Pi in biomineralization is underscored by a broad-range of mineralization defects in genetic and acquired disorders affecting systemic Pi homeostasis (hyperphosphatemia and hypophosphatemia) and local/cellular Pi availability (e.g. hypophosphatasia). Although it is well established now that Pi is a signaling molecule that can change cellular physiology, the underlying molecular mechanisms by which Pi executes this function remain largely unknown. This gap in our knowledge impedes development of targeted therapeutic approaches to diseases caused by abnormal Pi availability. Our goal is to delineate the signaling cascade that regulates biomineralization in response to extracellular Pi. We and others have shown that molecular interactions upstream of Erk1/2 kinase are central to initiating the response to extracellular Pi in the majority of analyzed cells. Our preliminary studies in cells producing mineralized extracellular matrix (osteogenic cells) identified a molecular circuit, which is required for activation of Erk1/2 and mineralization-supporting functions in response to Pi. First, our in vitro data show that osteogenic cells deficient in parathyroid hormone/parathyroid hormone related protein receptor 1 (Pth1r) do not activate Erk1/2 under high Pi conditions and have significantly impaired transcriptional response to Pi. Second, our data suggest that Pi-induced Erk1/2 activation and gene expression are dependent on the protein kinase C (PKC). Furthermore, our data suggest that the cellular response to Pi is enhanced by calcium (Ca2+). Based on these data we hypothesize that the Pi-induced signaling cascade is integrated with the Pth1r-PKC-Erk1/2 pathway in osteogenic cells. To test this hypothesis we will use in vitro and in vivo approaches focused on signaling in cells producing mineralized extracellular matrix. In the Aim 1 of this project, we will define the molecular circuit of Pth1r-dependent activation of cellular responses to Pi. In the Aim 2, we will determine the functional role of Pth1r in Pi signaling and Pi-regulated mineralization in vivo. In the Aim 3, we will determine the role of Ca2+ in mineralization-supporting functions of Pi signaling. By completing these Aims, will provide the first characterization of the Pi-induced signaling cascade and identify molecular players required for initiation of cellular responses to Pi in osteogenic cells. This will provide the foundation for identification of targets for pharmacological regulation of cellular sensitivity to available Pi.